A missense single-nucleotide polymorphism, C1858T in the PTPN22 gene is primarily associated with a wide range of human autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, and juvenile idiopathic arthritis. The PTPN22 gene encodes the lymphoid tyrosine phosphatase LYP, which is expressed only in white blood cells and acts as a gatekeeper of T lymphocyte activation. The molecular mechanism by which LYP tempers T lymphocyte activation involves the formation of a complex between LYP and the negative regulatory kinase Csk. We reported that the autoimmune-predisposing LYP-W620 variant cannot bind Csk, and more recently we found that the same variant is a gain-of-function form of the phosphatase. The increased inhibition of TCR signaling by LYP- W620 may lead to weaker signaling and therefore a failure to delete autoreactive T cells during thymic selection and/or insufficient activity of regulatory T cells. We hypothesize that a specific small-molecule inhibitor of LYP might be useful to revert the effects of LYP-W620 at the central and/or peripheral level, and prevent the emergence, or reappearance, of autoreactive T cells. Such an inhibitor would be widely applicable, both in helping to elucidate the biological role(s) of LYP and as a therapeutic in the treatment of many human autoimmune disorders. Unfortunately, there is currently no LYP assay available that is highly sensitive, continuous, and amenable to HTS. In the present proposal we will carry out all the preliminary work needed to begin a screening of NIH molecular libraries for inhibitors of LYP, and in particular we will (1) set up a reliable assay of LYP activity and (2) optimize it for HTS.